Proteolytic digestion of cardiac troponin i

ABSTRACT

A fluid sample container comprising a protease, when a sample fluid is placed in the fluid sample container the protease breaking a target analyte in the sample fluid into at least two peptides, the at least two peptides being smaller than the original target analyte.

The subject application claims benefit under 35 USC § 119(e) of U.S.Provisional Application No. 62/095,940, filed Dec. 23, 2014. The entirecontents of the above-referenced patent application are hereby expresslyincorporated herein by reference.

BACKGROUND 1. Field of the Disclosure

This disclosure relates to improving analytical recovery of analytesduring whole blood filtration and immunoassay analysis.

2. Brief Description of the Related Art

The inventive concepts described herein relate to a variety ofdiagnostic assays which seek to identify and qualify a target analyte ina medical patient's fluid sample.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 illustrates a cardiac troponin I (cTnI) molecule.

FIG. 2 illustrates an cTnI molecule that has been broken into more thanone peptides.

DETAILED DESCRIPTION OF THE INVENTIVE CONCEPT(S)

Before explaining at least one embodiment of the inventive conceptsdisclosed herein in detail, it is to be understood that the inventiveconcepts are not limited in their application to the details ofconstruction and the arrangement of the components or steps ormethodologies set forth in the following description or illustrated inthe drawings. The inventive concepts disclosed herein are capable ofother embodiments or of being practiced or carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein is for the purpose of description and should not beregarded as limiting the inventive concepts disclosed and claimed hereinin any way.

In the following detailed description of embodiments of the inventiveconcepts, numerous specific details are set forth in order to provide amore thorough understanding of the inventive concepts. However, it willbe apparent to one of ordinary skill in the art that the inventiveconcepts within the instant disclosure may be practiced without thesespecific details. In other instances, well-known features have not beendescribed in detail to avoid unnecessarily complicating the instantdisclosure.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a composition,a process, method, article, or apparatus that comprises a list ofelements is not necessarily limited to only those elements but mayinclude other elements not expressly listed or inherently presenttherein.

As used herein the terms “approximately,” “about,” “substantially” andvariations thereof are intended to include not only the exact valuequalified by the term, but to also include some slight deviationstherefrom, such as deviations caused by measuring error, manufacturingtolerances, wear and tear on components or structures, settling orprecipitation of cells or particles out of suspension or solution,chemical or biological degradation of solutions over time, stressexerted on structures, and combinations thereof, for example.

As used herein, the term “sample” and variations thereof is intended toinclude biological tissues, biological fluids, chemical fluids, chemicalsubstances, suspensions, solutions, slurries, mixtures, agglomerations,tinctures, slides, powders, or other preparations of biological tissuesor fluids, synthetic analogs to biological tissues or fluids, bacterialcells (prokaryotic or eukaryotic), viruses, single-celled organisms,lysed biological cells, fixed biological cells, fixed biologicaltissues, cell cultures, tissue cultures, genetically engineered cellsand tissues, genetically engineered organisms, and combinations thereof,for example.

Unless expressly stated to the contrary, “or” refers to an inclusive orand not to an exclusive or. For example, a condition A or B is satisfiedby anyone of the following: A is true (or present) and B is false (ornot present), A is false (or not present) and B is true (or present),and both A and B are true (or present). An inclusive or may beunderstood as being the equivalent to: at least one of condition A or B.

In addition, use of the “a” or “an” are employed to describe elementsand components of the embodiments herein. This is done merely forconvenience and to give a general sense of the inventive concepts. Thisdescription should be read to include one or at least one and thesingular also includes the plural unless it is obvious that it is meantotherwise.

As used herein, the term “fluid sample” and variations thereof isintended to include, for example, but not limited to, biological fluids(such as urine and whole blood), chemical fluids, chemical substances,suspensions, solutions, slurries, mixtures, agglomerations, tinctures,slides, or other preparations of biological fluids, synthetic analogs tobiological fluids, and combinations thereof.

Finally, as used herein any reference to “one embodiment,” “anembodiment,” or the like means that a particular element, feature,structure, or characteristic described in connection with the embodimentis included in at least one embodiment. The appearances of the phrase“in one embodiment” in various places in the specification are notnecessarily all referring to the same embodiment.

As will be appreciated by those skilled in the art, the inventiveconcepts described below can be applied to a variety of diagnosticassays which seek to identify and qualify a target analyte in a fluidsample. For purposes of this disclosure a target analyte should beunderstood as one or more of the following molecules: proteins, peptide,peptides, amino acids, and other types of amino acid analogues. Adiagnostic assay should be understood as a procedure by which the amountof a target analyte in the fluid sample is quantified. Typicaldiagnostic assays begin after a fluid sample has been obtained from apatient and placed in a fluid sample container. When the fluid sample iswhole blood this is typically performed by coupling the fluid samplecontainer to a syringe. One such fluid sample container for whole bloodis known as a “green top” sample tube and contains heparin—whichprevents the whole blood sample from clotting. Next, the fluid samplecan be inserted into a medical device (such as an analyzer) whichperforms the remaining steps of the assay. The medical device mayutilize a disposable insert (sometimes referred to as a consumable orcartridge) during the performance of assay. Alternatively, the fluidsample can be inserted into the disposable insert—which is then insertedinto the medical device.

In one embodiment of the invention the fluid sample is moved through afiltration device to arrive at a filtered fluid sample. This filtrationcan be performed using capillary action or a pumping means disposedwithin the medical device. In the case of whole blood, the filteroutputs plasma that is substantially free of Red Blood Cells (RBCs).Illustrative filtration devices include one or a combination of: afilter, a pre-filter, and/or an asymmetric membrane (ASM)—which containspores of varying sizes. Filtration device(s) may be disposed in themedical device, the disposable insert, or a combination of both. Oncefiltered, the amount of the target analyte in the filtered fluid sampleis determined through the appropriate use of antibodies, reagents, andelectrochemical sensors. In another embodiment of the invention, thefluid sample can be subjected to centrifugation.

An illustrative class of diagnostic assays relate to troponin. Intacttroponin in whole blood is an intracellular muscle protein with threesubunits [i.e., TnI (Inhibitory Subunit), TnT (Tropomyosin BindingSubunit) and TnC (Calcium Binding Subunit)]. It should be noted thatthere are specific subunits for cardiac muscle (i.e., cTnI and cTnT) butnot for TnC which is the same in all types of muscles.

An illustrative type of diagnostic assay relating to cardiac muscle is acardiac troponin (cTn) assay which determines the level of cTn proteinmolecules (i.e., the target analyte) in a fluid sample of whole blood.The detection of a rise and/or fall of cardiac troponin plays a key rolein the early diagnosis and management of myocardial infarction (MI) andacute coronary syndromes (ACS). Cardiac troponins, such as cardiactroponin I (cTnI) and cardiac troponin T (cTnT) protein molecules areproven biomarkers for the diagnosis and management of MI and ACS.Examples of cTn assays include, but are not limited to, cTnI assays,cTnT assays, and combined cTnI/cTnT assays—each of which targetdifferent cTn molecules or combinations of cTn molecules. A cTnI assaydetermines the level of cTnI molecules in a fluid sample of whole blood.A cTnT assay determines the level of cTnT molecules in a fluid sample ofwhole blood. A combined cTnI/cTnT assay determines the levels of cTnIand cTnT molecules in a fluid sample of whole blood. Certain cTn assaysmay also measure one ore more subunits of troponin (such as Tnl)—evenwhen that subunit is complexed to another subunit (such as TnT or TnC).

Like many other diagnostic assays, in current cTn assays a fluid sampleof whole blood is first obtained from a patient. Typically this sampleis obtained using a “green top” sample tube containing heparin—whichprevents the whole blood from clotting. Next, the whole blood isinserted into a medical device (such as an analyzer) and/or thedisposable insert. Upon receiving the fluid sample of whole blood, thewhole blood is filtered in order to arrive at plasma that issubstantially free from RBCs. Illustrative filtration devices removeRBCs from whole blood and include one or a combination of: a pre-filter,a filter, and/or an asymmetric membrane (ASM)—which contains pores ofvarying sizes. Once filtered, the amount of the target analyte (i.e.,cTnI, cTnT or cTnI/cTnT molecules) in the plasma is determined throughthe use of specific antibodies which bind to specific antibody bindregions (epitopes) of the target analyte (i.e., the cTnI, cTnT orcTnI/cTnT molecules). The resulting binding pairs can be detected by avariety of detections means based on the type of technology used in theassay—illustrative detection technology includes: fluorescence,chemiluminescence, electrochemical luminescence, electrochemically, andluminescent oxygen channeling immunoassay (“LOCI”).

While current whole blood filtration techniques are able to produce thedesired RBC free plasma, the inventors observed that the analyticalrecovery of cTnI and/or cTnT in current assays is low and inconsistent.One approach to improve recovery associated with non-specific binding isto pre-treat the filtration device with casein, sucrose and surfactantin an effort to improve the recovery of cTnI and/or cTnT from the fluidsample of whole blood. Another approach involves adding one of theanti-cTnI and/or cTnT antibodies used in the diagnostic assay to thefluid sample of whole blood sample prior to filtration. While theseapproaches have, to some degree, improved the analytical recovery of cTnassays, there is room for more improvement.

While working to improve the performance of cTn assays the inventorsobserved that a certain amount of cTn molecules were getting lost in theassay. For example, during a cTnI assay where the fluid sample of wholeblood had elevated hematocrit levels—e.g., levels where Red Blood Cells(RBCs) make up an unusually high percentage of the fluid sample of wholeblood—a significant percentage of cTnI was getting lost in the assay.This same phenomenon was also observed—to a lower extent—in the fluidsamples of whole blood with average or slightly above/below averagehematocrit levels. Without getting bound by theory, it is believed thatone or more, up to all, of the following factors contribute to the lossof cTnI: (1) the assay's filtration device itself could be filtering outcTnI; (2) cTnI could be sticking to the filtration device as it ispassing through; (3) RBCs that have accumulated in the assay'sfiltration device could be filtering out cTnI; and (4) the combinationof heparin (if present in the fluid sample of whole blood), RBCs andcTnI could be binding together—the combination of which may be gettingfiltered out by the assay's filtration device. One or more, up to all,of the factors could be responsible for the observed low recovery ofcTnI through the filtration device of an illustrative cTnI assay for agiven fluid sample of whole blood.

The inventive concepts disclosed herein improve the analytical recoveryof diagnostic assays during the filtration portion of the assay bybreaking the target analyte in a fluid sample into two or more peptidesthat are smaller, more well defined molecules than the original targetanalyte. Two, three or more peptides can be created from the targetanalyte by, for example, but not limited to, using a protease (i.e., aprotein that “digests” other proteins). By selecting an appropriateprotease, specific peptides can be created from the target analyte bybreaking the target analyte molecule at a precise location(s). As willbe explained below, the appropriate protease for a given target analytemolecule creates at least one diagnostically significant peptide thatfunctions with the underlying diagnostic assay and does not get caughtup in the filtration device. For purposes of this disclosure, a peptideshould be understood as containing a sequence of one or more aminoacids.

For example, in order to mitigate the four possible contributing factorsnoted above, a protease can be used to “break” the cTn into smaller,less “sticky,” molecules. By deliberately breaking the cTn molecules inthe proper places (as will be explained below) the resulting peptidesare (1) more likely to make it through the filtration device and (2)allow many, if not all, of the current cTn assays following whole bloodfiltration to properly function. Stated another way, by breaking cTnmolecule into peptides the inventive concepts disclosed herein haveimproved the recovery of cTn molecules in cTn assays without impactingthe underlying functionality of current cTn assays. These inventiveconcepts, therefore are compatible with most, if not all, cTn assayswhich use antibody bind regions (epitopes) of cTn molecules.

The separation of the target analyte (in this case cTn molecules) intopeptides can take place prior to, or contemporaneously with, filtrationof the fluid sample of whole blood. For example, the protease can bemixed with the fluid sample in the fluid sample container by placing theprotease in the fluid sample container before or after the fluid sampleis placed therein. In one example, a fluid sample container containingboth heparin and a protease may be provided to a medical professionalfor fluid sample collection. Alternatively, the liquid sample and theprotease may be mixed by the medical device prior to filtration orwithin the disposable insert. The protease can also be disposed withinthe filtration device so that the target analyte is broken into peptidesduring filtration.

The disposable insert discussed above may be single or multi use. Thedisposable insert contains compounds and/or components for use by themedical device during a diagnostic assay. For example, the disposableinsert can contain one or both of the protease and the filtrationdevice(s). An illustrative disposable insert includes, at least, asample fluid entry port, a protease reservoir, a chamber in which theprotease and fluid sample can be combined (which may be the proteasereservoir itself), a filtration device, an analysis chamber (where theoutput of the filtration device can be interrogated by the medicaldevice), and fluidic pathways connecting each of the aforementionedfeatures. An alternative illustrative disposable insert includes, atleast, a sample fluid entry port, a filtration device containing theappropriate amount of protease, an analysis chamber (where the output ofthe filtration device can be interrogated by the medical device), andfluidic pathways connecting each of the aforementioned features.

In various other embodiments, various aspect of the inventive aspectsdescribed herein may be performed by a combination of the medical deviceand the disposable insert in a wide variety of ways that should beapparent to those skilled in the art. In one embodiment, separation ofthe target analyte into peptides and filtration of the fluid sample cantake place in the medical device. In other embodiments, the separationof the target analyte into peptides and filtration of the fluid sampletakes place in the disposable insert. In another embodiment, thedisposable insert may contain the filtered sample for inspection by themedical device.

Using a cTnI molecule as an example of a cTn molecule, FIG. 1 depicts amap of the sequence of amino acids (shown by position numbers 1-210)which comprise a cTnI molecule 2. In FIG. 1, the following abbreviationsare used: A—Alanine, R—Arginine, N—Asparagine, D—Aspartic acid(Aspartate), C—Cysteine, Q—Glutamine, E—Glutamic acid (Glutamate),G—Glycine, H—Histidine, I—Isoleucine, L—Leucine, K—Lysine, M—Methionine,F—Phenylalanine, P—Proline, S—Serine, T—Threonine, W—Tryptophan,Y—Tyrosine, V—Valine, B—Aspartic acid or Asparagine, Z—Glutamine orGlutamic acid, and X—Any amino acid. Additionally, ‘TERM’ refers to atermination codon.

Continuing with FIG. 1, according to the inventive concepts disclosedherein, two, three or more peptides can be created from the cTnImolecule 2 by introducing a protease. By selecting an appropriateprotease, specific peptides 4 can be created from the cTnI molecule 2 bybreaking the cTnI molecule at a precise location(s). For example, thecTnI molecule can be broken into one, two, three, four, five, or morepeptides.

FIG. 2 illustrates a cTnI molecule 2 that has been broken into threepeptides 4, 6, and 8. In this embodiment, the cTnI was digested in orderto separate out the middle portion of the cTnI molecule (e.g., peptide6) which is the string of amino acids extending from approximatelyposition number 30 to position number 112. This middle portion 6 of thecTnI molecule is stable and contains enough of the characteristic of theoriginal cTnI molecule to allow existing antibody binding regions(epitopes) of certain cTnI assays to yield results—thus making it adiagnostically significant portion of the cTnI molecule. Based on thesequence of cTnI shown in FIG. 1, the illustrative protease chymotrypsincleaves proteins at aromatic hydrophobic amino acids (e.g., tyrosine—Y,tryptophan—W and phenylalanine—F) and yields proteolytic fragments(e.g., peptides 4 and 8) that leave the stable, middle portion (peptide6) of the cTnI molecule intact. Generally speaking a protease cleaves anamino acid at one or both of the C-Terminal side or the N-Terminal side.It is believed that the protease chymotrypsin cleaves on the C-Terminalside. In FIG. 2, the beginning of cTnI molecule 2 is broken off andrepresented by peptide 4 (position numbers 1 through 29) and the end ofcTnI molecule 2 is broken off and resented by peptide 8 (positionnumbers 113 through 210)—e.g., on the—Terminal of the tyrosine Y aminoacid. Heparin, which may be mixed with the fluid sample at some point inthe diagnostic assay, binds to RBCs and cTnI molecules between positionnumbers 113 and 210 (i.e. in peptide 8). By separating and allowingheparin to bind to peptide 8, rather than the entire molecule,diagnostically significant portions of the cTnI molecule (i.e., middlepeptide 6) are able to pass through the filtration device while thecombination of heparin, peptide 8, and RBCs gets filtered out. Whilepeptides 4 and 8 are shown as remaining intact, it should be appreciatedthat peptides 4 and 8 may be further broken down into smaller peptidesdepending on the protease used. Heparin binds to cTnT in a similarmanner—thus allowing for the diagnostically significant portion of thecTnT molecule to pass through the filtration device in a cTnT assay aswell.

It should be noted that chymotrypsin protease can also cleave proteinsat leucine—L and methionine—M amino acids residues (note that in thesequences shown in FIGS. 1 and 2 each amino acid is considered aresidue). This cleaving has the potential to break peptide 6 in FIG. 2into smaller peptides—which would negatively impact current assayresults. However, the the amount of cleaving at leucine—L andmethionine—M amino acid locations is notably lower than the cleavingwhich otherwise takes place at aromatic hydrophobic amino acids. Thus alarge enough number of diagnostically significant peptides 6 are formedto allow for most, if not all, of cTn assays to function properly.

According to one embodiment of the invention, the antibody is bound tothe cTnI molecule prior to proteolytic digestion to protect the epitope.When the antibody is bound to one of the epitopes it will prevent theprotease from digesting that portion of the amino acid sequence. Thisalso permits one of the binding events (the creation of theantibody:analyte complex) in the assay to be completed before or duringthe sample filtration process. It is possible that the protease coulddigest a portion of the antibody involved in the antibody:analytecomplex but is unlikely that complex would fall apart.

As should be appreciated by a person skill in the art, while the abovetext describes the inventive concepts in relation to cTn assays, theinventive concepts disclosed herein improve the recovery (as describedabove) are equally applicable to a wide variety of other diagnosticassays which target specific molecules that are prone to getting trappedin the assay's filtration device. Furthermore, the inventive conceptsdisclosed herein should also be understood as extending to diagnosticassays in which a filtration device does not contribute the loss of thetarget analyte. Possible reasons for this include instances where afilter is not present in the diagnostic assay or where a filter ispresent but does not filter, trap or otherwise prevent enough of thetarget analyte from passing through to have a measurable impact on thediagnostic assay itself. In any of these instances, certain diagnosticassays may still benefit from the use of a peptide to break the targetanalyte in a fluid sample into two or more peptides that are smaller,more well defined molecules than the original target analyte. Detectingthese smaller peptides may be, for example, easier or otherwise morereliable than detecting the target analyte itself.

The following is a non-limiting list of illustrative embodiments of theinventive concepts disclosed above:

1. A fluid sample container comprising: a protease, when a sample fluidis placed in the fluid sample container the protease breaking a targetanalyte in the sample fluid into at least two peptides, the at least twopeptides being smaller than the original target analyte.

2. The fluid sample container of illustrative embodiment 1 furthercomprising: heparin, when the sample fluid is whole blood the heparinpreventing red blood cells in the whole blood sample from clotting uponintroduction of the fluid sample into the fluid sample container, theheparin binding to less than all of the at least two peptides.

3. The fluid sample container of any of illustrative embodiments 1 to 2,wherein the protease is chymotrypsin and the target analyte is a cardiactroponin (cTn) molecule.

4. The fluid sample container of illustrative embodiment 3, wherein thecTn molecule is at least one of troponin I (cTnI) or cardiac troponin T(cTnT).

5. A device comprising: a sample fluid filter; and a protease, whereinwhen a sample fluid is mixed with the protease, the protease breaking atarget analyte in the sample fluid into at least two peptides, the atleast two peptides being smaller than the original target analyte.

6. The sample fluid filtration device of illustrative embodiment 5,wherein at least one of the at least two peptides is small enough topass through the sample fluid filtration device.

7. The device of illustrative embodiment 6, wherein the sample fluid isa whole blood sample; wherein the sample fluid further contains heparin,the heparin preventing red blood cells (RBCs) in the whole blood samplefrom clotting, the heparin binding to individual RBCs and to at leastone of the at least two peptides but less than all of the at least twopeptides to form a bound compound; and wherein the sample fluid filterprevents the bound compound from flowing there through.

8. The device of any of illustrative embodiments 5 to 7, wherein theprotease is disposed within the sample fluid filter.

9. The device of any of illustrative embodiments 5 to 7, wherein theprotease is mixed with the sample fluid in a sample container externalto the sample fluid filter.

10. The device of any of illustrative embodiments 5 to 7, wherein theprotease is chymotrypsin and the target analyte is a cardiac troponin(cTn) molecule.

11. The device of illustrative embodiment 10, wherein the cTn moleculeis at least one of troponin I (cTnI) or cardiac troponin T (cTnT).

12. A diagnostic assay comprising the steps of filtering a sample fluidmixture through a sample fluid filter, the sample fluid mixturecomprising a sample fluid and a protease, the protease breaking a targetanalyte in the sample fluid into at least two peptides that are smallerthan the original target analyte, the at least two peptides comprisingat least a first peptide and a second peptide, the sample fluid filterallowing at least the first peptide to flow there through and preventingat least the second peptide from flowing there through to produce afiltered sample fluid mixture; and determining an amount of the firstpeptide in the filtered sample fluid mixture.

13. The diagnostic assay of illustrative embodiment 12, furthercomprising: prior to filtering, receiving the sample fluid mixture.

14. The diagnostic assay of illustrative embodiment 13, furthercomprising: prior to filtering, receiving the sample fluid; and prior tofiltering but after receiving, mixing the sample fluid with the proteaseto create the sample fluid mixture.

15. The diagnostic assay of any of illustrative embodiment 12 to 14,wherein the sample fluid is whole blood and further contains heparin,the heparin preventing red blood cells (RBCs) in the whole blood samplefrom clotting, the heparin binding to individual RBCs and to at leastthe second peptide but not the first peptide to form a bound compound;and wherein the sample fluid filter prevents the bound compound fromflowing there through.

1.-4. (canceled)
 5. A device comprising: a sample fluid filter; and aprotease, wherein when a heparinized whole blood sample is mixed withthe protease, the protease breaking a cardiac troponin in theheparinized whole blood sample into at least two peptides, the at leasttwo peptides being smaller than the original target analyte; wherein atleast one of the at least two peptides is small enough to pass throughthe sample fluid filtration device; and wherein the heparin prevents redblood cells (RBCs) in the whole blood sample from clotting, the heparinbinding to individual RBCs and to at least one of the at least twopeptides but less than all of the at least two peptides to form a boundcompound; and wherein the sample fluid filter prevents the boundcompound from flowing there through. 6.-7. (canceled)
 8. The device ofclaim 5, wherein the protease is disposed within the sample fluidfilter.
 9. The device of claim 5, wherein the protease is mixed with thesample fluid in a sample container external to the sample fluid filter.10. The device of claim 5, wherein the protease is chymotrypsin.
 11. Thedevice of claim 10, wherein the cTn molecule is at least one of cardiactroponin I (cTnI) or cardiac troponin T (cTnT). 12.-15. (canceled) 16.The device of claim 5, wherein the cardiac troponin is cardiac I (cTnI)and is broken down into at least three peptides.
 17. The device of claim16, wherein at least one of the middle peptide fragments of cTnI has theamino acid sequence represented by SEQ ID NO:3.
 18. The device of claim16, wherein the heparin present in the whole blood sample fluid binds toa C-terminal peptide fragment of cTnI and wherein the C-terminal peptidefragment of cTnI has the amino acid sequence represented by SEQ ID NO:4.19. The device of claim 17, wherein the middle peptide fragment isdetected using a detection technology selected from fluorescence,chemiluminescence, electrochemical luminescence, electrochemically, andluminescent oxygen channeling immunoassay (LOCI).
 20. The device ofclaim 16, wherein the at least three peptides comprises at least anN-terminal peptide fragment, a C-terminal peptide fragment, and a middlepeptide fragment, wherein the heparin present in the whole blood samplefluid binds to the C-terminal peptide fragment of cTnI; and wherein theN-terminal peptide fragment of cTnI has the amino acid sequencerepresented by SEQ ID NO:2.